Early development of the mammalian retina proceeds through a series of cellular changes in which progenitor cells exit mitosis, migrate to particular laminar positions and terminally differentiate into one of seven basic neural or glial cell types. Defects in these events lead to human eye malformations, including optic nerve aplasia or hypoplasia, congenital glaucoma and cone/rod dystrophic syndromes. The genetic control of these events is poorly understood at present. However, recent findings suggest that key proteins controlling some of these developmental processes are nuclear transcription factors. This application will use embryological, genetic, immunohistochemical, histological and molecular methods to explore the function of the mouse Math5 gene. Math5 encodes a basic helix-loop-helix transcription factor whose expression in retinal progenitors foreshadows the terminal birthdates of retinal ganglion cells (RGCs). In preliminary studies we have created a targeted deletion of the Math5 gene and analyzed the postnatal phenotypes in the retina. Math5 -/- adult mice are fully viable but lack optic nerves, chiasmata and RGCs. An increase in cone photoreceptor cells was also observed. The aims of this application will further investigate the role of Math5 during eye development. Although postnatal mutant eyes contain no RGCs, we have observed a great reduction, but not complete loss, of differentiating RGCs in mutant embryos. These findings will be further investigated using gene expression studies and the creation of chimeric mutant embryos through aggregation chimera production. A new expression domain for Math5 has also been identified in the forming optic stalk. The identity of these optic stalk cells and a potential function for Math5 during optic stalk development will be studied. The hierarchical relationships of Math5 and other eye pattern formation (Pax6, Hes1, Pax2, Chx10) and retinal neuron (Math3, Mash 1, Ngn2, NeuroD, Brn3b, Isl-1) genes will be explored through gene expression and mutational analyses. Comparison of the genomic structure and sequence of Math5 and its human orthologue, Hath5, demonstrates the presence of eight highly conserved genomic sequence elements located 5' and 3' to the coding region. These sequences may represent the gene promoter and tissue-specific expression control elements. In vitro and in vivo experiments will test whether these sequences regulate Math5 gene expression. The proposed studies should yield valuable new information concerning the pathogenesis of eye disease and may provide a foundation for better diagnosis and prevention of these diseases in the future.